Vesicle Compositions

ABSTRACT

Vesicle compositions are provided that comprise a therapeutic compound. The vesicle compositions may be capable of releasing the therapeutic compound in response to the presence of an external trigger. The vesicle compositions may comprise a plurality of biocompatible vesicles. The biocompatible vesicles may comprise a therapeutic compound for treatment of a patient in need thereof, and one or more cross-linkages between two or more of the biocompatible vesicles, each cross-linkage comprising a chemical sensing moiety and a sensed moiety. In some embodiments, the therapeutic compound may be any compound that provides palliative, curative, or otherwise beneficial effects to a patent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. patent application Ser. No.13/411,415, filed on Mar. 2, 2012, and U.S. Provisional PatentApplication No. 61/448,556, filed on Mar. 2, 2011, each of which areentirely incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under R43 DK083819awarded by the National Institute of Health. The Government has certainrights in the invention.

BACKGROUND

Diabetes is a disease caused by an insulin deficiency or a resistance toinsulin in the body. Type I diabetes is an autoimmune disease thatdamages the beta cells of the islets of Langerhans in the pancreas,resulting in an inadequate amount of insulin in the body. Withoutinsulin treatments, Type I diabetes is fatal. Type II diabetes resultsfrom an insufficient production of insulin or an inability of thepatient's body to respond properly to insulin. Insulin resistanceassociated with Type II diabetes prevents adequate levels of blood sugarfrom entering into cells to be stored for energy, resulting inhyperglycemia in the bloodstream. Traditionally, Type I diabetes istreated by repeated subcutaneous injections of insulin each day. Type IIdiabetes is also treated with insulin, often in combination with othermedications taken orally or by injection. Multiple injections of insulinper day are required, as well as careful monitoring of the patient'sblood glucose levels through dietary control and blood testing. Insulinpumps are used as an alternative to multiple daily insulin injections bya syringe. However, insulin pumps are costly, must be worn most of thetime, and require blood testing to determine the amount of insulin todeliver into the patient. Blood testing requires the patient to draw asample of blood, usually from a finger, and to test the blood sample forglucose concentration. Blood glucose monitoring systems are availablethat use a sensor placed just under the skin to periodically monitor theamount of glucose in the interstitial fluid. These systems requirecalibration, typically resulting in two finger pricks per day, and arecostly. Moreover, a lag time exists between the concentration of glucosein the bloodstream and the concentration of glucose in the interstitialfluid.

A need exists for a system or device that delivers controlled amounts ofinsulin directly in response to increased glucose concentrations,without requiring the patient or medical professional to continuouslymonitor the blood glucose level, determine the appropriate amount ofinsulin to be injected, and inject the insulin periodically throughoutthe day.

SUMMARY

Vesicle compositions are provided that comprise a therapeutic compound.The vesicle compositions may be capable of releasing the therapeuticcompound in response to the presence of an external trigger. The vesiclecompositions may comprise a plurality of biocompatible vesicles. Thebiocompatible vesicles may comprise a therapeutic compound for treatmentof a patient in need thereof, and one or more cross-linkages between twoor more of the biocompatible vesicles, each cross-linkage comprising achemical sensing moiety and a sensed moiety. In some embodiments, thetherapeutic compound may be any compound that provides palliative,curative, or otherwise beneficial effects to a patent. The vesiclecompositions may be suitable for injection into a patient.

Vesicle compositions are also provided that comprise a plurality ofbiocompatible vesicles, the biocompatible vesicles comprising atherapeutic compound for treatment of a patient in need thereof, and oneor more cross-linkages between two or more of the biocompatiblevesicles, each cross-linkage comprising a boronic acid moiety or aboronic acid derivative moiety and a sugar moiety.

Further, methods are provided for administering a therapeutic compoundto a patient in need thereof. These methods may comprise injecting avesicle composition parenterally into a patient in need of treatment,the vesicle composition comprising a therapeutic compound; and releasingthe therapeutic compound into the patient in response to a triggeringevent.

Methods are also provided for treating a medical condition, the methodscomprising administering a vesicle composition into a patient in need oftreatment, wherein the vesicle composition is loaded with a therapeuticcompound for treatment of a patient in need thereof; and releasing thetherapeutic compound into the patient in response to a triggering event.

The general description and the following detailed description areexemplary and explanatory only, and are not intended to be restrictiveof the invention, as defined in the claims. Other embodiments willbecome apparent to those skilled in the art, in view of the detaileddescription provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying figures, chemical formulas, chemical structures, andexperimental data are given that, together with the detailed descriptionprovided below, describe example embodiments of the claimed invention.

FIG. 1 illustrates one embodiment of a vesicle composition.

FIG. 2 illustrates one embodiment of a vesicle composition.

FIG. 3 illustrates one embodiment of a vesicle composition.

FIG. 4 shows representative images of HeLa cells showing the effect ofboronic acid derivative treatment on the nuclear or cytoplasmiclocalization of NF-κB.

FIG. 5A demonstrates the Pearson's correlation coefficient (PCC) betweenthe nuclear and cytoplasmic fractions of the NF-κB molecule in HeLacells treated with free (dark bar) 4-aminocarbonylphenylboronic acid, aswell as each of those boronic acid derivates as conjugates ofDSPE-PEG-COOH (light bar), as compared to an untreated control (UTC) andlipopolysaccharide (LPS).

FIG. 5B demonstrates the Pearson's correlation coefficient (PCC) betweenthe nuclear and cytoplasmic fractions of the NF-κB molecule in HeLacells treated with free (dark bar) 3-aminophenylboronic acid, as well aseach of those boronic acid derivates as conjugates of DSPE-PEG-COOH(light bar), as compared to an untreated control (UTC) andlipopolysaccharide (LPS).

FIG. 5C demonstrates the Pearson's correlation coefficient (PCC) betweenthe nuclear and cytoplasmic fractions of the NF-κB molecule in HeLacells treated with free (dark bar) 5-amino-2,4-diflourophenylboronicacid, as well as each of those boronic acid derivates as conjugates ofDSPE-PEG-COOH (light bar), as compared to an untreated control (UTC) andlipopolysaccharide (LPS).

FIG. 5D demonstrates the Pearson's correlation coefficient (PCC) betweenthe nuclear and cytoplasmic fractions of the NF-κB molecule in HeLacells treated with free (dark bar) 3-fluoro-4-aminomethylphenylboronicacid, as well as each of those boronic acid derivates as conjugates ofDSPE-PEG-COOH (light bar), as compared to an untreated control (UTC) andlipopolysaccharide (LPS).

FIG. 5E demonstrates the Pearson's correlation coefficient (PCC) betweenthe nuclear and cytoplasmic fractions of the NF-κB molecule in HeLacells treated with free (dark bar) and 4-aminopyrimidineboronic acid, aswell as each of those boronic acid derivates as conjugates ofDSPE-PEG-COOH (light bar), as compared to an untreated control (UTC) andlipopolysaccharide (LPS).

FIG. 6A demonstrates the surviving fractions of HeLa cells treated withfree (dark bar) 4-aminocarbonylphenylboronic acid, as well as each ofthose boronic acid derivates as conjugates of DSPE-PEG-COOH (light bar),as compared to a UTC and LPS.

FIG. 6B demonstrates the surviving fractions of HeLa cells treated withfree (dark bar) 3-aminophenylboronic acid, as well as each of thoseboronic acid derivates as conjugates of DSPE-PEG-COOH (light bar), ascompared to a UTC and LPS.

FIG. 6C demonstrates the surviving fractions of HeLa cells treated withfree (dark bar) 5-amino-2,4-diflourophenylboronic acid, as well as eachof those boronic acid derivates as conjugates of DSPE-PEG-COOH (lightbar), as compared to a UTC and LPS.

FIG. 6D demonstrates the surviving fractions of HeLa cells treated withfree (dark bar) 3-fluoro-4-aminomethylphenylboronic acid, as well aseach of those boronic acid derivates as conjugates of DSPE-PEG-COOH(light bar), as compared to a UTC and LPS.

FIG. 6E demonstrates the surviving fractions of HeLa cells treated withfree (dark bar) 4-aminopyrimidineboronic acid, as well as each of thoseboronic acid derivates as conjugates of DSPE-PEG-COOH (light bar), ascompared to a UTC and LPS.

FIG. 7 shows a representative plot for the binding constant calculationfor 4-aminocarbonylphenylboronic acid.

FIG. 8 shows the structure, glucose binding affinity, percent HeLa cellsurvival at 80 nM, and the PCC between the nuclear and cytoplasmicfractions of the NF-κB molecule in HeLa cells at 80 nM, of variousboronic acid derivatives.

FIG. 9 illustrates cumulative release plots for four representativeboronic acid-sugar vesicle compositions.

FIG. 10A illustrates cumulative plots for release of insulin from4-aminocarbonylphenylboronic acid-sugar vesicle compositions triggeredwith 5 mM, 7 mM, and 10 mM glucose, as compared to triggering with 5 mM,7 mM, and 10 mM PBS.

FIG. 10B illustrates differential plots for release of insulin from4-aminocarbonylphenylboronic acid-sugar vesicle compositions triggeredwith 5 mM, 7 mM, and 10 mM glucose, as compared to triggering with 5 mM,7 mM, and 10 mM PBS.

FIG. 11A illustrates cumulative plots for release of insulin from4-aminocarbonylphenylboronic acid-sugar vesicle compositions(“4-aminocarbonylphenylboronic acid AVT”) and Concanavalin A vesiclecompositions (“ConA-AVT”) triggered with 10 mM, 30 mM, and 40 mMglucose.

FIG. 11B illustrates differential plots for release of insulin from4-aminocarbonylphenylboronic acid-sugar vesicle compositions(“4-aminocarbonylphenylboronic acid AVT”) and Concanavalin A vesiclecompositions (“ConA-AVT”) triggered with 10 mM, 30 mM, and 40 mMglucose.

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to thefollowing detailed description taken in connection with the accompanyingfigures and examples, which form a part of this disclosure. Thisinvention is not limited to the specific devices, methods, applications,conditions, or parameters described and/or shown herein. The terminologyused herein is for the purpose of describing particular embodiments byway of example only, and is not intended to be limiting. As used in thespecification and the claims, the singular forms “a,” “an,” and “the”include the plural. The term “plurality,” however, means more than one.Reference to a particular numerical value includes at least thatparticular value, unless the context clearly dictates otherwise. When arange of values is expressed, another embodiment includes from the oneparticular value and/or to the other particular value. The particularvalue forms another embodiment. Where the term “about” is used inconjunction with a number, it is intended to include ±10% of the number.For example, “about 10” may mean from 9 to 11.

To the extent that the term “includes” or “including” is used in thespecification or the claims, it is intended to be inclusive in a mannersimilar to the term “comprising” as that term is interpreted whenemployed as a transitional word in a claim. Furthermore, to the extentthat the term “or” is employed (e.g., A or B) it is intended to mean “Aor B or both.” When the applicants intend to indicate “only A or B butnot both” then the term “only A or B but not both” will be employed.Thus, use of the term “or” herein is the inclusive, and not theexclusive use.

Certain features of the invention which are, for clarity, describedherein in the context of separate embodiments, may also be provided incombination in a single embodiment. Conversely, various features of theinvention that are, for brevity, described in the context of a singleembodiment, may also be provided separately or in any subcombination.

The present invention may be more readily understood by reference toFIGS. 1, 2, and 3. In FIG. 1, a schematic of one embodiment of a vesiclecomposition 1 is shown. As shown in FIG. 1, vesicle composition 1comprises a plurality of biocompatible vesicles 2. In one embodiment,the plurality of biocompatible vesicles 2 may be, for example, non-toxicand biodegradable. As shown in FIG. 1, vesicle composition 1 furthercomprises one or more chemical sensing moieties 3. In one embodiment,the one or more chemical sensing moieties 3 may be, for example, one ormore chemical moieties that are capable of reversibly bonding with aconjugate moiety. As shown in FIG. 1, vesicle composition 1 furthercomprises one or more sensed moieties 4. In one embodiment, the one ormore sensed moieties 4 may be, for example, chemical moieties that arecapable of reversibly bonding with a chemical sensing moiety, e.g., 3.As used herein, the phrase “sensed moiety” may encompass both boundsensed moieties that are attached to a biocompatible vesicle (as shownin FIG. 1), and free sensed moieties that are sensed moieties not boundto a biocompatible vesicle but are present in the physiologicalenvironment (i.e., the environment within the body of a patient).Returning to FIG. 1, a first biocompatible vesicle 2 is attached to achemical sensing moiety 3, while a second biocompatible vesicle 2 isattached to a sensed moiety 4. Chemical sensing moiety 3 and sensedmoiety 4, when attached together, form crosslink 5.

FIG. 2 illustrates another embodiment of a vesicle composition 1. Afirst biocompatible vesicle 2 is attached to a polymer linker 6, and thepolymer linker 6 is attached to a chemical sensing moiety 3. A secondbiocompatible vesicle 2 is attached to a sensed moiety 4. The chemicalsensing moiety 3 and sensed moiety 4, when attached together, formcrosslink 5.

FIG. 3 illustrates still another embodiment of a vesicle composition 1.A biocompatible vesicle 2 is attached to a first polymer linker 6, andthe first polymer linker 6 is attached to a first chemical sensingmoiety 3. A second biocompatible vesicle 2 is attached to a secondpolymer linker 6, and the second polymer linker 6 is attached to asecond chemical sensing moiety 3. Two sensed moieties 4 are attachedtogether and each chemical sensing moiety 3 is attached to a sensedmoiety 4. The chemical sensing moieties 3 and sensed moieties 4, whenattached together, form a crosslink 5.

A biocompatible vesicle may be any biocompatible particle capable ofcarrying a therapeutic compound. The biocompatible vesicle may beapproximately spherical in shape with an inner portion. The therapeuticcompound can be carried in the inner portion of the vesicle, in the wallof the vesicle, attached to the outer surface of the vesicle, or by anyother suitable means. The biocompatible vesicle may comprise polymers,lipids, proteins, carbohydrates, other macromolecules, waters, andsalts, or any combination thereof. For example, the biocompatiblevesicle may comprise a liposome comprised of a plurality of lipids. Thelipids may comprise saturated lipids. In one embodiment, thebiocompatible vesicle may be comprised ofdistearoylphosphatidylethanolamine (DSPE),dipalmitoylphosphatidylethanolamine (DPPE),dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), or any combination thereof. The biocompatible vesicle may have asufficient shelf life to be suitable as a delivery system for atherapeutic compound. In one embodiment, the biocompatible vesicle mayhave a shelf life stability of at least six months at from about 2° C.to about 8° C., or a shelf life stability of at least seven days at roomtemperature.

Suitable vesicle compositions may include a therapeutic compound. In oneembodiment, the therapeutic compound may be loaded in or on thebiocompatible vesicles. In one embodiment, the vesicle composition maybe capable of releasing the therapeutic compound into the physiologicalenvironment of a patient. The physiological environment of the patientis ordinarily the bloodstream, but may be any environment within thepatient's body into which the vesicle composition is delivered. In oneembodiment, the therapeutic compound will be released from the vesiclecomposition in response to the presence of a free sensed moiety in thephysiological environment. In one embodiment, the free sensed moiety inthe physiological environment that triggers release of the therapeuticcompound is symptomatic of the condition sought to be treated by thetherapeutic compound. In formulations where the vesicle is suitable forinjection into a patient, the weight percentage of the therapeuticcompound based on the total weight of the vesicle composition may be inthe range of from about 0.1% to about 30%, or from about 0.2% to about20%, or from about 1% to about 10%. The therapeutic compound may be anycompound administered to a patient to provide palliative, curative, orotherwise beneficial effects. Example therapeutic compounds may includeinsulin and ciprofloxacin. In some embodiments where the therapeuticcompound is insulin, the insulin may be present in a concentration inthe range of from 0.1 mg/mL to 10 mg/mL, or from about 0.5 mg/mL toabout 5 mg/mL, or from about 1 mg/mL to 3 mg/mL, such as 2 mg/mL.

In some embodiments, the quantity of therapeutic compound released isrelated to the quantity of sensed moiety present in the physiologicalenvironment of the patient. For example, the quantity of therapeuticcompound released and the quantity of sensed moiety in the environmentmay be linearly proportional or may be related by a non-linear function.Accordingly, an increase in the concentration of free sensed moiety inthe physiological environment may trigger an increase in the amount oftherapeutic compound released from the vesicle composition. For example,the vesicle composition may release the therapeutic compound insulin inresponse to the presence of free sensed moieties that are sugars in thephysiological environment. When the concentration of sugar free sensedmoieties increases, the concentration of insulin released may alsoincrease. Example sugars may include glucose, galactose, maltose,lactose, fructose, sucrose, or any combination thereof. In oneembodiment, the release of insulin may be triggered by the presence ofglucose in the physiological environment. In alternative embodiments,sugars may be used to trigger the release of therapeutic compounds otherthan insulin.

In some embodiments, the chemical sensing moiety may be attacheddirectly to the biocompatible vesicle. A linker moiety can be positionedbetween the chemical sensing moiety and the biocompatible vesicle. Inembodiments where the biocompatible vesicle is a liposome, the linkermoiety may be attached to a lipid that is part of the liposome. Thelinker moiety may be a biodegradable polymer moiety, such as, forexample, polyethylene glycol (PEG). The linker moiety may provideflexibility between the biocompatible vesicle and the chemical sensingmoiety. One way of characterizing the flexibility of the linker moietyis the density of unsaturated bonds in each repeating unit of thepolymer moiety. In one embodiment, each repeating unit of the polymercomprising the linker moiety may contain at least one unsaturated bond.Another way of characterizing the flexibility of the linker moiety is byits length. Where the linker moiety is PEG, each PEG moietyindependently may have a molecular weight in the range of from about 100to about 10,000 Da, such as, for example, in the range of from about 500to about 5,000 Da. In one embodiment, the PEG linker moiety comprisesabout 10-100 ethylene glycol repeating units. In another embodiment, thePEG linker moiety comprises about 30-60 ethylene glycol repeating units.

The sensed moiety may be capable of being attached, such as by acovalent bond, to the chemical sensing moiety. The sensed moiety may bea bound sensed moiety that is attached to a biocompatible vesicle,which, together with a chemical sensing moiety, forms a crosslink in thevesicle composition. Alternatively, the sensed moiety may be a freesensed moiety, which competes with bound sensed moieties to bond with achemical sensing moiety. In one embodiment, the free sensed moiety inthe physiological environment may serve as a trigger to releasetherapeutic compound by competitively bonding with a chemical sensingmoiety and thereby cleaving crosslinks between biocompatible vesicles.

The sensed moiety may be related to the disease or condition sought tobe treated by the therapeutic compound. For example, in some embodimentswhere the condition sought to be treated by the therapeutic compound isdiabetes or another metabolic disorder that results in hyperglycemia,the free sensed moiety that triggers release of the therapeutic compoundis a sugar. Example sugars may include glucose, galactose, maltose,lactose, fructose, sucrose, or any combination thereof. In oneembodiment, the sugar is glucose. In embodiments where the condition tobe treated is diabetes or another metabolic disorder that results inhyperglycemia and the free sensed moiety is a sugar, the therapeuticcompound may be insulin. In some embodiments where the condition soughtto be treated is inflammation, the chemical compound that triggers therelease of the therapeutic compound may be nitric oxide. In someembodiments, amyloid beta 42 may trigger the release of a therapeuticcompound to treat potential plaque formation. In other embodiments,albumin, alkaline phosphatase, alanine transaminase (ALT), aspartateaminotransferase (AST), blood urea nitrogen (BUN), calcium, chloride,carbon dioxide, creatinine, direct bilirubin, gamma-glutamyltranspeptidase (Gamma-GT), glucose, lactate dehydrogenase (LDH),phosphorus, potassium, sodium, total bilirubin, total cholesterol, totalprotein, uric acid, or any combination thereof may act as a free sensedmoiety.

The chemical sensing moiety and the sensed moiety are chemical groupsthat may be capable of reversibly attaching to each other. Theattachment may be a chemical bond, such as a covalent bond. The covalentbond between the chemical sensing moiety and the sensed moiety may becapable of cleaving in the presence of competing free sensed moieties inthe environment. When the vesicle composition has been delivered into apatient, the covalent bond between the chemical sensing moiety and thesensed moiety may be capable of cleaving in the presence of free sensedmoieties in the physiological environment. Where different chemicalsensing moieties are attached to each biocompatible vesicle, thecrosslinkages formed by the different chemical sensing moieties and thesensed moieties may have different strengths. The degree of cleavage ofcrosslinks and the rate of release of the therapeutic compound maydepend on the number of strong, weak, and moderate crosslinkagespresent.

Suitable chemical sensing moieties may include a boronic acid or aboronic acid derivative. Example boronic acid derivatives may includephenylboronates, pyridylboronates, and cyclohexylboronates. In oneembodiment, the boronic acid derivative may be3-(N,N-dimethylamino)phenyl boronic acid; 2,4-dichlorophenylboronicacid; 4-aminocarbonylphenylboronic acid; 3-chlorophenylboronic acid;4-hydroxyphenylboronic acid; 4-propylphenylboronic acid;3-[(E)-2-nitrovinyl)phenylboronic acid; 4-chlorocarbonylphenylboronicanhydride; cyclopenten-1-ylboronic acid; 2-bromopyridine-3-boronic acid;2,4-ditert-butoxypyrimidin-5-ylboronic acid;2,4-bis(benzyloxy)pyrimidine-5-boronic acid; 5-phenyl-2-thienylboronicacid; 5-formylthiophene-3-boronic acid; or any combination thereof

The sensed moiety may be a sugar in embodiments in which the chemicalsensing moiety is a boronic acid or a boronic acid derivative. Suitablesugars may include glucose, galactose, maltose, lactose, fructose,sucrose, or any combination thereof. In one embodiment, the sensedmoiety may be glucose where the chemical sensing moiety is boronic acidor a boronic acid derivative. In some embodiments, each biocompatiblevesicle may have only one kind of boronic acid or boronic acidderivative attached to it. In other embodiments, each biocompatiblevesicle may have either one kind of boronic acid or boronic acidderivative attached to it or more than one kind of boronic acid attachedto its surface. Where different boronic acid derivatives are attached toeach biocompatible vesicle, the crosslinkages formed by the differentboronic acid derivatives may have different strengths. The degree ofcleavage of crosslinks and the rate of release of the therapeuticcompound may depend on the number of strong, weak, and moderatecrosslinkages present.

The biocompatible vesicles, chemical sensing moieties, sensed moieties,and crosslinks forming the vesicle composition may be arranged in anynumber of ways. The biocompatible vesicles may each have multiplemoieties attached, the moieties being chemical sensing moieties, sensedmoieties, or both. Alternatively, the biochemical vesicles each may haveonly one moiety attached, either a chemical sensing moiety or a sensedmoiety. Also, the biocompatible vesicles each may have any number ofmoieties attached with some biocompatible vesicles having one moietyattached and other biocompatible vesicles having more than one moietyattached. In some embodiments, the biocompatible vesicles may have bothchemical sensing moieties and sensed moieties attached to the samebiocompatible vesicle. In one embodiment, the biocompatible vesicleseach have only chemical sensing moieties or only sensed moietiesattached. Suitable vesicle compositions may be comprised of as few astwo biocompatible vesicles. Suitable vesicle compositions may alsocomprise between about 10 and about 10⁸ biocompatible vesicles. In oneembodiment, the vesicle composition may comprise between about 10² andabout 10⁷ biocompatible vesicles. In one embodiment, the vesiclecomposition may comprise between about 10³ and about 10⁷ biocompatiblevesicles. The moieties attached to the biocompatible vesicles may bearranged in any geometry that will allow crosslinks to be formed andcleaved between chemical sensing moieties and sensed moieties. Thechemical sensing or sensed moieties may be arranged on the biocompatiblevesicle in any geometry. In some embodiments, the moieties will beequally spaced about the biocompatible vesicle. In other embodiments,moieties will be attached to the biocompatible vesicle in a randomfashion. Regardless of the geometry of the biocompatible particles,attached moieties, and crosslinks which they form, the vesiclecomposition may assume a folded or otherwise agglomerated geometry. Insome embodiments, the vesicle composition may have a diameter in therange of from about 0.1 micrometers to about 50 micrometers. In oneembodiment, the vesicle composition may have a diameter in the range offrom about 0.5 micrometers to about 30 micrometers. In one embodiment,the vesicle composition may have a diameter in the range of from about 1micrometer to about 20 micrometers.

The vesicle composition may be suitable for injection into a patient.One method for measuring suitability for injection is to test for thedegree of inflammation caused by a PEG-lipid composition comprising thechemical sensing moiety. One measure for the degree of inflammation isan NF-κβ protein assay. In one embodiment, the PEG-lipid compositioncomprising the chemical sensing moiety tested using an NF-κβ assayresults in a PCC of less than about 0.2. In one embodiment, when theconcentration of the PEG-lipid composition comprising the chemicalsensing moiety is in the range of from about 40 nM to 160 nM, e.g.,about 80 nM, the PCC between the nuclear and cytoplasmic fraction of theNF-κβ molecule is less than about 0.2, e.g., less than about 0.1. In oneembodiment, the PEG-lipid composition comprising the chemical-sensingmoiety may be capable of causing less inflammation than the chemicalsensing moiety single molecule, as measured by an NF-κβ translocationassay.

Another method for measuring the suitability for injection of thevesicle composition is to measure the cell toxicity of the chemicalsensing moiety. The vesicle composition is suitable for injection if thechemical sensing moiety is characterized as giving rise to greater thanabout 82% cell survival as measured according to an MTT cellproliferation assay. The chemical sensing moiety may be characterized asgiving rise to greater than 82% cell survival as measured according toan MTT cell proliferation assay, when the chemical sensing moiety isdosed at a concentration in the range of from about 40 nM to about 160nM.

In another embodiment, a method is provided for administering atherapeutic compound to a patient in need thereof, the method comprisinginjecting a vesicle composition parenterally into the patient, thevesicle composition including a therapeutic compound, and releasing thetherapeutic compound into the patient in response to a triggering event.The vesicle composition may be comprised of a therapeutic compound,biocompatible vesicles, one or more chemical sensing moieties, one ormore sensed moieties, and crosslinks between the chemical sensing andsensed moieties. In one embodiment, the triggering event is symptomaticof the condition sought to be treated by the therapeutic compound. Inone embodiment, the triggering event may be the presence of free sensedmoiety in the physiological environment. In some embodiments, anincrease in concentration of free sensed moiety in the physiologicalenvironment may result in release of therapeutic compound.

In one embodiment, the method may comprises administering a vesiclecomposition where the chemical sensor is a boronic acid or boronic acidderivative, the sensed moiety is a sugar, and the therapeutic compoundis insulin. In one embodiment, boronic acid derivatives may includephenylboronates, pyridylboronates, and cyclohexylboronates. In oneembodiment, the boronic acid derivative may be3-(N,N-dimethylamino)phenyl boronic acid; 2,4-dichlorophenylboronicacid; 4-aminocarbonylphenylboronic acid; 3-chlorophenylboronic acid;4-hydroxyphenylboronic acid; 4-propylphenylboronic acid;3-[(E)-2-nitrovinyl)phenylboronic acid; 4-chlorocarbonylphenylboronicanhydride; cyclopenten-1-yl boronic acid; 2-bromopyridine-3-boronicacid; 2,4-ditert-butoxypyrimidin-5-ylboronic acid;2,4-bis(benzyloxy)pyrimidine-5-boronic acid; 5-phenyl-2-thienylboronicacid; 5-formylthiophene-3-boronic acid; or any combination thereof. Inembodiments in which the chemical sensing moiety is a boronic acid or aboronic acid derivative, the sensed moiety may be a sugar. Examplesugars may include glucose, galactose, maltose, lactose, fructose,sucrose, or any combination thereof. In one embodiment, where thechemical sensing moiety is boronic acid or a boronic acid derivative,the sensed moiety is glucose. In some embodiments, each biocompatiblevesicle has only one kind of boronic acid or boronic acid derivativeattached to it. In other embodiments, each biocompatible vesicle haseither one kind of boronic acid or boronic acid derivative attached toit or more than one kind of boronic acid attached to it. Where differentboronic acid derivatives are attached to each biocompatible vesicle, thecrosslinkages formed by the different boronic acid derivatives may havedifferent strengths. The degree of cleavage of crosslinks and the rateof release of the therapeutic compound may depend on the number ofstrong, weak, and moderate crosslinkages present.

In one embodiment, the triggering event may be hyperglycemia within thepatient. In some embodiments, an increase in the glucose concentrationin the physiological environment in the patient may trigger release ofthe therapeutic compound. In other embodiments, an increase in the sugarconcentration in the physiological environment in the patient maytrigger an increase in therapeutic compound released from the vesiclecomposition. In still other embodiments, the presence of sugar in thephysiological environment may trigger the release of therapeuticcompound in a quantity proportional to that of the sugar. In oneembodiment, the sugar may be glucose, galactose, maltose, lactose,fructose, sucrose, or any combination thereof. In one embodiment, thesugar is glucose. In some embodiments, the triggering event may be whenthe sugar concentration in the physiological environment in the patientis greater than about 100 mg/dL.

In one embodiment, the vesicle composition may be administered betweentwice in one day to once a week, for an indefinite duration of days. Inone embodiment, the vesicle composition may be administered once perday, for an indefinite duration of days. In some embodiments, thevesicle composition may be further characterized as being comprised ofagglomerated vesicles.

In one embodiment, a method is provided for treating a medicalcondition, the method comprising administering a vesicle compositioninto a patient in need of treatment, the vesicle composition beingloaded with a therapeutic compound for treatment of a patient in needthereof, and releasing the therapeutic compound into the patient inresponse to a triggering event. The vesicle composition may comprisebiocompatible vesicles, one or more chemical sensing moieties, one ormore sensed moieties, and crosslinks between the chemical sensing andsensed moieties. The vesicle composition may comprise an agglomerationof biocompatible vesicles. The vesicle composition may be administeredby any suitable means, including, for example, by injection.Alternatively, the vesicle composition may be administered by pump orinhalation. In one embodiment, the vesicle composition may beadministered between twice in one day to once a week, for an indefiniteduration of days. In one embodiment, the vesicle composition may beadministered once per day, for an indefinite duration of days.

Methods described herein may include administering a vesicle compositionwhere the chemical sensor is a boronic acid or boronic acid derivative,the sensed moiety is a sugar, and the therapeutic compound is insulin.Boronic acid derivatives may include phenylboronates, pyridylboronates,and cyclohexylboronates. In one embodiment, the boronic acid derivativemay comprise 3-(N,N-dimethylamino)phenyl boronic acid;2,4-dichlorophenylboronic acid; 4-aminocarbonylphenylboronic acid;3-chlorophenylboronic acid; 4-hydroxyphenylboronic acid;4-propylphenylboronic acid; 3-[(E)-2-nitrovinyl)phenylboronic acid;4-chlorocarbonylphenylboronic anhydride; cyclopenten-1-yl boronic acid;2-bromopyridine-3-boronic acid; 2,4-ditert-butoxypyrimidin-5-ylboronicacid; 2,4-bis(benzyloxy)pyrimidine-5-boronic acid;5-phenyl-2-thienylboronic acid; 5-formylthiophene-3-boronic acid; or anycombination thereof. In embodiments in which the chemical sensing moietyis a boronic acid or a boronic acid derivative, the sensed moiety maycomprise a sugar. Example sugars may include glucose, galactose,maltose, lactose, fructose, sucrose, or any combination thereof. In oneembodiment where the chemical sensing moiety is boronic acid or aboronic acid derivative, the sensed moiety comprises glucose. In someembodiments, each biocompatible vesicle may have only one kind ofboronic acid or boronic acid derivative attached to it. In otherembodiments, each biocompatible vesicle may have either one kind ofboronic acid or boronic acid derivative attached to it or more than onekind of boronic acid attached to it. Where different boronic acidderivatives are attached to each biocompatible vesicle, thecrosslinkages formed by the different boronic acid derivatives may havedifferent strengths. The degree of cleavage of crosslinks and the rateof release of the therapeutic compound may depend on the number ofstrong, weak, and moderate crosslinkages present.

Vesicle compositions may also include a targeting mechanism. Thetargeting mechanism may be any method of directing the vesiclecomposition to a specific destination within the patient's body. In oneembodiment, the targeting mechanism may be a cell receptor, an antibody,a biomarker, or any combination thereof. In some embodiments, thevesicle composition may include a contrast agent, a diagnostic agent, orboth.

A number of medical conditions may be treated by the inventive methods,including metabolic disorders. Examples of metabolic disorders mayinclude any medical condition related to the body's metabolism,especially diabetes. A therapeutic compound for treating diabetes (e.g.,insulin and its variants) is capable of sustaining normoglycemic levelsfor 24-72 hours. A normoglycemic level of sugar in the bloodstream istypically about 126 mg/dL.

The methods of the invention may also be used to treat pulmonaryinfection. When the methods of the invention are treating pulmonaryinfection, the therapeutic compound may be an antibiotic. The antibioticmay be any antibiotic suitable to treat pulmonary infection. Inembodiments were the medical condition being treated is pulmonaryinfection, the therapeutic compound may be, for example, ciprofloxacin.

EXAMPLES Example 1 NF-κB Assay for Inflammation

The inflammatory potential of various boronic acid compounds was studiedby measuring the nuclear translocation of NF-κB in HeLa cells byimmunocytochemistry and high throughput high contact microscopy. 15,000HeLa cells were plated in 96 well plates the night before theexperiments. On the day of the experiments, the cells were treated withthe boronic acids (Table 1, below) at three different concentrations (40nM, 80 nM, and 160 nM) for 2 h. At the end of the incubation, cells werewashed with phosphate buffered saline (PBS) and were fixed in 4%paraformaldehyde for 15 min and permeablized with 0.01% Triton X-100 inPBS for 10 min. The cells were washed with PBS three times. Nonspecificsites were blocked with 5% bovine serum albumin (BSA) in PBS andincubated with the Anti-NF-κB for 1 h, followed by incubation withfluorescein isothiocyanate (FITC)-labeled secondary antibody. Afterwashing the cells at the end of the incubation, the cells were treatedwith 4′,6-diamidino-2-phenylindole (DAPI) for 1 min and kept at 4° C.until further analysis. Images of the cells were acquired with aBeckman-Coulter 100 automated high-throughput microscope system andanalyzed using Cytseer software (Vala Sciences, CA). The co-localizationof NF-κB was quantified by measuring the PCC between the nuclear andcytoplasmic fraction of the NF-κB molecule. The PCC is a measure of theoverlap between the pixel intensities of the nuclear and NF-κB images ofthe same cell. The PCC values can range from −1 to 1. A positivecorrelation (PCC value) indicates nuclear translocation of NF-κB. Anegative correlation (negative PCC value) indicates an absence ofnuclear translocation.

FIG. 4 shows representative images of HeLa cells illustrating the effectof boronic acid treatment on the nuclear or cytoplasmic localization ofNF-κB. DAPI is shown by white circles, while NF-κB is represented bylight gray. White circles surrounded by light gray indicate cytoplasmicNF-κB, while obliteration of the white circles by the gray signal in thenucleus indicates nuclear NF-κB. (A) UTC, cytoplasmic NF-κB; (B)Positive control, nuclear translocation of NF-κB in cells treated withLPS; (C) Treatment with 2,4-di(tert-butoxy)pyrimidine-5-yl-boronic acid(80 nM, 2 hrs), predominantly cytoplasmic NF-κB; and (D) Treatment with5-isoquinolineboronic acid (80 nM, 2 hrs.), predominantly nuclear NF-κB.

FIG. 5 demonstrates the PCC between the nuclear and cytoplasmicfractions of the NF-κB molecule in HeLa cells treated with free (darkbar) 4-aminocarbonylphenylboronic acid, 3-aminophenylboronic acid,5-amino-2,4-diflourophenylboronic acid,3-fluoro-4-aminomethylphenylboronic acid, and 4-aminopyrimidineboronicacid, as well as each of those boronic acid derivates as conjugates ofDSPE-PEG-COOH (light bar), as compared to a UTC and LPS. Surprisingly,the 4-aminocarbonylphenylboronic acid conjugate was less inflammatorythan free 4-aminocarbonylphenylboronic acid.

Table 1 and FIG. 8 list the PCC results for various boronic acidderivatives.

TABLE 1 Sugar Binding Affinity MTT (% Cell Survival) Inflammation (PCC)Log(Ka_(BA)/ 40 nM 80 nM 160 nM 40 nM 80 nM 160 nM Ka_(ConA))2-bromopyridine-5-boronic acid 95.1013 120.589 35.78268877 0.00604 0.0780.144556 3-(N,N-dimethylamino)phenyl 123.37 86.2247 85.91160221 −0.1236−0.0401 0.186204 −0.14 boronic acid 2-napthylboronic acid 24.383137.2192 21.10497238 0.06402 0.24369 0.0959313-carboxy-5-nitrophenylboronic 54.3094 46.2431 33.29650092 −0.01360.03893 0.122719 acid uracil-5-boronic acid 68.5083 50.3499 28.06629834−0.0586 0.00516 −0.015223 2-chloro-3-quinolineboronic acid 30.110544.7882 32.43093923 0.08644 0.05095 0.192957 2-propoxyphenylboronic acid45.7459 42.2099 41.95211786 −0.0398 0.03035 −0.0495392-cyanophenylboronic acid 117.42 62.63 60.33 −0.0641 0.0475 0.1042,4-dicholorophenylboronic acid 99.29 81.59 79.9 −0.0502 0.09386 0.3067−0.93 thianthrene-1-boronic acid 64.19 61.32 81.10 −0.0683 0.02680.109448 cyclopropylboronic acid 81.55 107.79 80.01 −0.0484 0.034750.170472 2,6-dimethoxyphenylboronic 92.66 78.05 48.60 −0.0679 0.038880.260667 acid 3-aminocarbonylphenylboronic 101.02 77.93 29.19 −0.06960.07022 0.174824 acid 3-(benzyloxy)phenylboronic acid 99.83 71.94 28.92−0.1159 0.0809 0.376808 3-nitrophenylboronic acid 58.14 55.55 38.03−0.0801 0.06937 0.233508 4-cyanophenylboronic acid 60.40 63.69 54.22−0.0705 −0.204 0.487922 4-aminocarbonylphenylboronic 86.57 97.67 57.28−0.0838 0.01211 0 −1.03 acid 3-cyano-4-fluorophenylboronic 89.30 60.4647.56 −0.0231 0.10144 0 acid 2-chloropyridine-4-boronic acid 84.93 58.4756.36 −0.1195 0.00374 0 3-hydroxyphenylboronic acid 104.60 62.75 89.29−0.1136 0.04005 0 trans-2-Phenylvinylboronic acid 72.90 99.51 65.87−0.1303 0.15234 0 4-chlorophenylboronic acid 51.23 81.65 56.97 −0.14940.01664 0 1-phenylvinylbornic acid 66.08 67.23 58.91 −0.1145 0.03776 02-thienylboronic acid 53.48 75.76 61.19 −0.121 −0.0343 04-(dimethylamino)phenylboronic 78.59 71.75 51.04 −0.1318 −0.00510.212957 acid 1-(triisopropylsilyl)pyrrol-E-3- 72.92 65.92 56.94 −0.07810.00445 0.16903. boronic acid 5-bromopyridine-3-boronic acid 134.74837.2764 30.03715319 −0.038 0.13112 0.291325 3-chlorophenylboronic acid97.1779 73.129 38.55244882 −0.0824 0.03311 0.396508 −1.03N-methylindole-2-boronic acid 63.3131 63.6075 47.46038056 −0.126 0.007460.133554 N-boc-2-pyrroleboronic acid 60.6873 51.264 39.41134031 −0.06750.05743 0.183701 2-hydroxyphenylboronic acid 70.3314 55.8039 53.64439929−0.0198 −0.0878 0.088843 2-formyl-3-thiopheneboronic 55.3376 40.368446.20885296 −0.0993 −0.0793 0.126198 acid 2-chlorophenylboronic acid86.3068 60.0002 45.4481205 −0.0957 .00949 0.0994774-hydroxyphenylboronic acid 203.926 198.184 172.8339984 −0.0665 −0.10810.200707 −1.08 4-propylphenylboronic acid 202.674 202.92 164.1960039−0.1402 −0.0786 0.192226 −0.009 5-isoquinolineboronic acid 75.80 60.8441.69 −0.0412 0.02463 0.278406 5-fluoro-2- 84.22 84.22 54.92 −0.01690.02474 0.293611 methoxyphenylboronic acid pentafluorophenylboronic acid65.63 60.00 50.98 0.0154 0.0762 0.258448 5-formyl-2-furanboronic acid77.18 94.08 50.14 0.00989 −0.0122 0.113246 5-acetyl-2-thiopheneboronicacid 84.50 82.81 51.27 0.02454 0.06312 0.23431 3-thienylboronic acid90.42 80.00 49.85 −0.0062 0.14569 0.059629 5-cyanothiophene-2-boronicacid 80.56 40.00 44.78 −0.01 0.10511 0.243354-ethylsulfonyl-phenylboronic 67.61 66.47 37.46 0.00184 0.1802 0.129047acid 5-bromobenzo-B-thophene- 71.83 81.40 90.42 −0.0244 −0.01 0.1265272boronic acid 2-ethoxy-5-pyridine-boronic acid 81.40 69.58 33.80 −0.1416−0.0136 0.149833 2-(N,N- 74.92 76.38 47.32 −0.1529 −0.0404 0.034217dimethylsulphamoyl)benzene- boronic acid 3-bromothiophene-4-boronic76.45 95.90 97.49 −0.0695 0.15676 0.152756 acid3-bromothiophene-5-boronic 116.67 97.63 62.08 −0.1162 −0.023 0.153863acid 3-[(E)-2- 116.00 96.38 73.93 −0.0456 −0.0517 0.205842 NAnitrovinyl]phenylboronic acid 5-formylthiophene-3-boronic 86.87 87.0373.80 −0.0359 0.00225 0.193025 −1.24 acid 4-chlorocarbonylphenylboronic114.54 107.21 79.46 −0.0664 −0.0012 0.098262 −2.08 anhydridecyclopenten-1-yl boronic acid 99.21 79.90 68.16 −0.0551 0.01168 0.163417NA 2-bromopyridine-3-boronic acid 102.96 94.00 87.88 −0.0553 −0.03960.149396 −2.9 2,4-di-tert-butoxypyrimidin-5- 100.46 90.60 71.81 −0.08880.03106 0.172566 0.008 ylboronic acid 2,4-bis(benzyloxy)pyrimidine-5-100.05 89.04 74.74 −0.0647 −0.0533 0.04709 NA boronic acid3-formylfuran-2-boronic acid 107.84 73.72 65.18 −0.0012 0.0274 0.0862135-phenyl-2-thienylboronic acid 100.56 81.24 86.23 −0.0211 −0.07010.118672 −2.3 1-benzyl-1H-pyrazole-4-boronic 100.88 62.54 86.11 −0.1081−0.0841 0.03839 acid 4-dibenzofuranboronic acid 71.48 75.50 70.50−0.0736 −0.0438 −0.127966 3,5-dimethylisoxazole-4-boronic 88.37 90.6342.89 −0.061 0.0057 −0.012768 acid 5-cyano-2- 81.55 49.84 62.08 −0.0808−0.0523 −0.080857 methoxyphenylboronic aciddiisopropyl(bromomethyl)borona 81.27 96.62 79.32 −0.0205 0.02739 0.11569te

Example 2 MTT Assay for Cytotoxicity

The cytotoxicity of the boronic acid derivatives from Table 1 wasstudied by MTT assay. 150,000 HeLa cells were plated in 96 well plateson the night before the experiments. On the day of experiments, thecells were treated with the boronic acid derivatives at three differentconcentrations (40 nM, 80 nM, and 160 nM) for 2 h. At the end of theincubation, MTT assays (In-Vitro toxicology MTT based assay kit, SigmaAldrich, MO) were performed according to the manufacturer's protocol.

FIG. 6 demonstrates the surviving fractions of HeLa cells treated withfree (dark bar) 4-aminocarbonylphenylboronic acid, 3-aminophenylboronicacid, 5-amino-2,4-diflourophenylboronic acid,3-fluoro-4-aminomethylphenylboronic acid, and 4-aminopyrimidineboronicacid, as well as each of those boronic acid derivates as conjugates ofDSPE-PEG-COOH (light bar), as compared to a UTC and LPS. Surprisingly,the 4-aminocarbonylphenylboronic acid conjugate was less cytotoxic thanfree 4-aminocarbonylphenylboronic acid.

Table 1 and FIG. 8 list the cell survival rate for various boronic acidderivatives.

Example 3 Binding Assay

The binding affinity of boronic acid derivatives to glucose wasdetermined by competition assay using ConcanavalinA (ConA) as thecompetitive standard, and varying concentrations of the boronic acidderivatives. Carboxy-terminated magnetic beads were activated bysuspending the beads in 100 mM 2-(N-morpholino)ethanesulfonic acid (MES)buffer (pH 4.5). Glucosamine (100 mg/mL) was conjugated to the activatedmagnetic beads using carbodiimide coupling with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as a crosslinker.The conjugation reaction was carried out in a 96 well plate. Thereaction was run for 24 h. The beads were separated by placing themicroplate on a magnetic separator and were washed thoroughly with PBSat pH 7.2 to remove unbound glucose, excess EDC, andN-hydroxysulfosuccinimide (Sulfo-NHS).

The glucose terminated beads were co-incubated for 1 h with 2.4 μM ConA(a known binder of carbohydrates) fluorescently tagged with fluoresceinisothiocyanate (ConA-FITC), and titrated with concentrations rangingfrom 2.5 μM to 20 μM of each boronic acid derivative tested. Thefollowing controls were used: (A) carboxy-terminated magnetic beads (notconjugated to glucose) were treated with ConA-FITC to determinenon-specific binding; and (B) glucose conjugated beads were treated witha high concentration of non-fluorescent ConA to determine maximalbinding.

The wells were washed three times with PBS at pH 7.2 to remove unboundboronic acid derivatives and FITC-ConA. The beads were re-suspended inPBS and stirred for 15 min at rt before measuring FITC fluorescence(excitation: 495 nm, top emission: 520 nm; 6 flashes per well; averageof n=3 wells in a Flexstation 11³⁸⁴ microplate reader to quantify theamount of glucose bound to the boronic acids). The fluorescenceintensity was measured and used as an indication of the amount of ConAbound to the surface of the beads, or [ConAs]. The ConA-sugarinteraction was expected to be inhibited by the boronic acidderivatives, which can bind the sugar molecules and displace the boundConA-FITC, thereby causing a decrease in fluorescence intensity of themixture.

The binding constant of the boronic acid derivatives was calculated fromthe fluorescence intensity. The reduction of fluorescence intensity isrelated to [ConAs] by Equation 2.

$\begin{matrix}{\lbrack {ConA}_{s} \rbrack = {( {1 - \frac{F_{\max} - F_{obs}}{F_{\max}}} )*\lbrack {ConA}_{s} \rbrack}} & ( {{Eq}.\mspace{14mu} 2} )\end{matrix}$

The relationship between [ConAs] and the amount of boronic acid [BA] isgiven by Equation 3.

$\begin{matrix}{\lbrack {ConA}_{s} \rbrack = \frac{S_{t}.\lbrack{ConA}\rbrack}{1 + \frac{\lbrack{BA}\rbrack \cdot {Ka}_{ConA}}{\lbrack{ConA}\rbrack \cdot {Ka}_{BA}}}} & ( {{Eq}.\mspace{14mu} 3} )\end{matrix}$

Simple rearrangement yields Equation 4, from which the ratio of the twoequilibrium constants (K_(ConA)/K_(BA)) can be derived.

$\begin{matrix}{\frac{1}{\lbrack {ConA}_{s} \rbrack} = {\frac{1}{S_{t}} + {\frac{\lbrack{BA}\rbrack}{\lbrack{ConA}\rbrack S_{t}}*\frac{{Ka}_{ConA}}{{Ka}_{BA}}}}} & ( {{Eq}.\mspace{14mu} 4} )\end{matrix}$

-   -   The concentration of sugar sites (S1) can be calculated from the        intercept of a plot of 1/[ConAs] vs 1/[BA].

FIG. 7 shows a representative plot for the binding constant calculationfor 4-aminocarbonylphenylboronic acid.

Table 1 lists the relative sugar binding affinities(log(K_(ConA)/K_(BA))) of various boronic acid derivatives, compared toConA.

FIG. 8 shows the structure and glucose binding affinity (M⁻¹) forvarious boronic acid derivatives.

Example 4 Synthesis of Lipid-PEG-Linker-Boronic Acid Conjugates

All lipid-PEG-linker-boronic acid and sugar conjugates were prepared bycoupling the amine-derivative of the boronic acid-linker or sugarmoieties, with DSPE-PEG-COOH using carbodiimide chemistry. In general,50 mg of DSPE-PEG-COOH were dissolved in 2 mL anhydrousdimethylformamide (DMF), followed by the addition of EDC (2.0equivalents) and N-Hydroxysuccinimide (NHS) (3.0 equivalents), and themixture was allowed to stir for 30 min at rt. The amine derivative ofthe boronic acid or sugar was added and the reaction mixture was allowedto stir overnight. For amines obtained in the form of the ammonium salt,these were de-salted by stirring in 500 μL DMF and 35 μL triethylaminefor 30 min at rt prior to addition to the reaction mixture. The reactionmixture was diluted with 4 mL MES buffer (50 mM, pH 4.18), transferredinto a 3K MWCO dialysis cassette, and dialyzed twice against 2 L of thesame buffer, then twice against 2 L water, followed by freeze drying toobtain the desired conjugate. The identity and purity of the finalproduct was confirmed by ¹H NMR spectrometry.

Example 5 Synthesis of Liposomes Functionalized with Sugar/Boronic Acidsand their Conjugates

Synthesis of phospholipid-polyethylene glycol-gyclosyl conjugates.Lipid-PEG-Glycosyl species were synthesized by conjugating the carboxylgroup of DSPE-PEG-COOH to the amino group of glucosamine, galactosamine,and mannopyranoside, using the carbodiimide coupling chemistry describedin G.T. Hermanson, Bioconjugate Techniques, Academic Press, ElsevierScience USA (1996) 169-185, incorporated herein by reference. Thecoupling was done at the C2 position of the sugar molecules, therebykeeping the C3, C4, and C5 positions unmodified.

Loading of liposomes with insulin. Human recombinant insulin wasdissolved in citrate buffer (100 mM, pH 2.5) to a concentration of 15mg/mL. Lipids (56.4 mole % DPPC, 40 mole % cholesterol, and 1.2 mole %each of DSPE-PEG-Glucose, DSPE-PEG-Galactose, DSPE-PEG-Mannopyranoside)were dissolved in ethanol and hydrated with the insulin solution at 50°C. for 15 min. The final lipid concentration was 50 mM. The hydratedmixture was passed eight times through a 400 nm Nucleopore track-etchmembrane at 50° C. at a pressure of 100 psi. The parent liposomes had amean diameter of 244.1 nm (suitable average liposome diameters may befrom about 100 nm to about 300 nm) and a lipid concentration of about 50mM. Insulin was encapsulated by passive loading. The pH of the liposomalformulation was maintained at 5.6 (isoelectric point of insulin). Theliposomes were dialyzed against citrate buffer (100 nM, pH 5.6) toremove the unencapsulated insulin. In this example, unencapsulatedinsulin is removed to mitigate or prevent the insulin from having animmediate and, perhaps, unnecessary effect upon the physiologicalenvironment. In some embodiments, the vesicle composition is intended torelease and/or provide insulin primarily or solely in response to thepresence of a threshold amount of glucose in the physiologicalenvironment of the patient (i.e., having a “glucose responsive” effect),such as when the patient is experiencing a hyperglycemic condition. Inthe instant example, the encapsulated insulin was present in aconcentration of approximately 15 mg/mL, although it is contemplatedthat the concentration of the encapsulated insulin may be approximatelythe same as, or may be less than or greater than, the startingconcentration of insulin. The unencapsulated insulin may be present in aconcentration of approximately 0%-5% of the encapsulated insulin, or upto about 0.75 mg/mL, although higher concentrations of unencapsulatedinsulin may be acceptable or preferred, depending on the patient's needand the medium by which the vesicle composition is introduced into thephysiological environment of the patient.

Synthesis of phospholipid-polyethylene glycol-boronic acid derivativeconjugates. Lipid-PEG-boronic acid was synthesized by conjugating thecarboxyl group of DSPE-PEG-COOH to the amino functionalized boronic acidmoieties using the carbodiimide coupling chemistry described in G. T.Hermanson, Bioconjugate Techniques, Academic Press, Elsevier Science USA(1996) 169-185, incorporated herein by reference. The lipid compositionfor the boronic acid functionalized liposome was the following: 56.4mole % DPPC, 40 mole % cholesterol, and 3.6 mole % of DSPE-PEG-boronicacid.

Representative lipid-PEG-boronic acid derivative conjugate speciesinclude:

3-Aminophenylboronic acid conjugate. Characteristic ¹H NMR peaks: δ ppm7.98 (s, 1H), 7.59 (d, 1H), 7.31 (d, 1H), 7.03 (t, 1H), 5.33 (s, 1H,NH), 5.21 (s, 2H, NH), 1.55 (t, 3H), 1.49 (t,3H).

3-Fluoro-4-aminomethylphenylboronic acid conjugate. Characteristic ¹HNMR (300 MHz, acetone-d6) peaks: δ ppm 8.15 (d, 1H), 8.00 (dd, 1H), 7.90(m, 1H), 5.70 (s, 1H, NH), 5.50 (s, 1H, NH), 1.55 (t, 3H), 1.40 (t, 3H).

4-Aminopyrimidylboronic acid conjugate. Characteristic ¹H NMR (300 MHz,acetone-d6) peaks: δ ppm 7.85 (s, 2H), 5.25 (s, 1H, NH), 5.20 (s, 2H,NH), 1.40 (t, 6H).

4-Aminocarbonylphenylboronic acid conjugate. Characteristic ¹H NMR (300MHz, acetone-d6) peaks: δ ppm 7.85 (d, 2H), 7.80 (d, 2H), 5.30 (s, 1H,NH), 5.25 (s, 2H, NH), 1.40 (t, 6H).

5-Amino-2,4-difluorophenylboronic acid.

The insulin loading, extrusion, and purification processes were similarto that used for the sugar liposomes described above. The liposome had amean diameter of 184±0.162 nm (suitable average liposome diameters maybe from about 100 nm to about 300 nm). The encapsulated insulin waspresent in a concentration of approximately 15 mg/mL.

Preparation of vesicle compositions. Two kinds of insulin loadedliposomal formulations, functionalized either with sugar molecules orboronic acid derivatives, were mixed and stirred at rt to form vesiclecompositions. The mixtures were prepared using several mole ratios ofsugar species to boronic acid species on the external surface of theliposomes, ranging from 1:2 to 1:50, to determine the excess of boronicacids required for vesicle composition formation. The pH of the mixturewas also varied between 7 and 11, in order to select vesiclecompositions that are formed at physiological pH.

TABLE 2 Example Conditions for Preparing Boronic Acid-Sugar VesicleCompositions Sugar Liposome:Boronic Boronic Acid Moiety Acid Liposome(v/v) pH 3-aminophenylboronic acid 1:5  7.55-amino-2,4-difluorophenylboronic acid 1:5  84-aminocarbonylphenylboronic acid 1:20 83-fluoro-4-aminomethylphenylboronic acid 1:10 8

Confirmation of chemical cross-linkage of boronic acid and sugarmoieties. To confirm that the vesicle compositions were formed bychemical cross-linking of the boronic acid and sugar moieties, theagglomerates were exposed to 10 mM glucose. The agglomerates werereadily cleaved in the presence of glucose, due to competitive bindingof the boronic acids with the free glucose. This was demonstrated by theincrease in frequency of particles sized under 1 μm from 13% to 37% uponincubation with glucose.

Example 6 In-Vitro Release of Insulin from the Boronate-Sugar VesicleCompositions

A small volume (500 μL) of the agglomerates was loaded inside a tubulardialysis membrane (100,000 MWCO), sealed with clips, and dialyzedagainst a PBS solution (pH 7.4) for 30 min prior to cleaving withglucose to monitor the passive diffusion of insulin without trigger.This was followed by addition of glucose solution to the agglomeratesinside the membrane at regular intervals to cleave the vesiclecompositions and trigger the release of insulin. Aliquots were removedfrom the external phase every 15 min, and the insulin concentration wasassayed by reading the absorbance at 214 nm. The assay was continued forseveral hours to verify a halt in release of encapsulated insulin.

FIG. 9 illustrates cumulative release plots for four differentboronate-glucose vesicle compositions, (4-aminocarbonylphenylboronicacid, 3-aminophenylboronic acid, 3-fluoro-4-aminoethylphenylboronicacid, and 5-amino-2,4-difluorophenylboronic acid), in the absence ofglucose trigger. The 3-aminophenylboronic acid vesicle composition andthe 4-aminocarbonylphenylboronic acid vesicle composition released 12%and 17% of their insulin content, respectively.

Insulin release from the 4-aminocarbonylphenylboronic acid vesiclecomposition upon addition of varying concentrations of glucose triggerwas assayed for several hours. The trigger concentrations were chosen tomimic blood glucose levels at normoglycemic and hyperglycemicconditions. A control assay was also performed where the4-aminocarbonylphenylboronic acid vesicle composition was triggered withPBS instead of glucose. The 4-aminocarbonylphenylboronic acid vesiclecomposition exhibited a burst release of insulin within two min afterintroduction of the glucose trigger. The release rate slows down overtime until another dose of glucose is added, which triggered a newepisode of burst release. In this example, the minimum concentration ofglucose required for cleaving the 4-aminocarbonylphenylboronic acidvesicle composition and releasing insulin was 10 mmol/L, whichcorresponds to a blood glucose level of 180 mg/dL. No burst release ofinsulin was observed when the 4-aminocarbonylphenylboronic acid vesiclecomposition was triggered with glucose concentrations analogous tohypoglycemia (about 9 mg/dL) or normoglycemia (about 126 mg/dL),demonstrating that the example 4-aminocarbonylphenylboronic acid vesiclecomposition is suitable for maintaining normal blood glucose levels.Also, the insulin release rate from these vesicle compositions isdependent on the glucose concentration, thereby making the presentembodiments useful in mitigating or avoiding hyper-insulinism.

FIG. 10 illustrates (a) cumulative and (b) differential plots forrelease of insulin from 4-aminocarbonylphenylboronic acid vesiclecompositions (4-aminocarbonylphenylboronic acid AVT) triggered with 5mM, 7 mM, and 10 mM glucose, as compared to triggering with 5 mM, 7 mM,and 10 mM PBS.

FIG. 11 illustrates (a) cumulative and (b) differential plots forrelease of insulin from 4-aminocarbonylphenylboronic acid and ConAvesicle compositions triggered with 10 mM, 30 mM, and 40 mM glucose.

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the embodiments disclosed herein, and thatsuch changes and modifications can be made without departing from thespirit of the invention. It is, therefore, intended that the appendedclaims cover all such equivalent variations as fall within the truespirit and scope of the invention.

What is claimed is:
 1. A vesicle composition, comprising: a firstliposome, comprising: a first phospholipid; cholesterol; and aphospholipid-polyethylene glycol-boronic acid derivative conjugate; asecond liposome, comprising: a second phospholipid; cholesterol; and aphospholipid-polyethylene glycol-glycosyl conjugate, at least one of thefirst liposome or the second liposome respectively encapsulating a firsttherapeutic compound or a second therapeutic compound; and thephospholipid-polyethylene glycol-boronic acid derivative conjugate ofthe first liposome and the phospholipid-polyethylene glycol-glycosylconjugate of the second liposome together being configured to form atleast one reversible boronic-glycosyl crosslink between the firstliposome and the second liposome, provided that when: a glycosyl moietyof the phospholipid-polyethylene glycol-glycosyl conjugate comprises aglucosyl moiety, a galactosyl moiety, or a mannopyranosidyl moiety; thefirst liposome encapsulates the first therapeutic compound; the secondliposome encapsulates the second therapeutic compound; and the firstliposome is coupled to the second liposome by the at least onereversible boronic-glycosyl crosslink, the phospholipid-polyethyleneglycol-boronic acid derivative conjugate is not represented by

wherein n=30-60.
 2. The vesicle composition of claim 1, comprising theat least one reversible boronic-glycosyl crosslink between thephospholipid-polyethylene glycol-boronic acid derivative conjugate ofthe first liposome and the phospholipid-polyethylene glycol-glycosylconjugate of the second liposome.
 3. The vesicle composition of claim 1,the phospholipid-polyethylene glycol-boronic acid derivative conjugatecomprising a 4-aminocarbonylphenylboronic acid moiety.
 4. The vesiclecomposition of claim 1, the phospholipid-polyethylene glycol-boronicacid derivative conjugate being represented by:

wherein n=30-60.
 5. The vesicle composition of claim 1, the glycosylmoiety of the phospholipid-polyethylene glycol-glycosyl conjugatecomprising one of: the glucosyl moiety, the galactosyl moiety, or themannopyranosidyl moiety.
 6. The vesicle composition of claim 1, thefirst liposome encapsulating the first therapeutic compound and thesecond liposome encapsulating the second therapeutic compound.
 7. Thevesicle composition of claim 6, the first therapeutic compound and thesecond therapeutic compound being the same.
 8. The vesicle compositionof claim 6, the first therapeutic compound and the second therapeuticcompound each comprising insulin.
 9. The vesicle composition of claim 1,at least one of the first and the second liposomes being configured torelease an amount of at least one of the corresponding first and secondtherapeutic compounds in response to a presence of a sugar.
 10. Thevesicle composition of claim 9, the amount released of at least one ofthe corresponding first and second therapeutic compounds being dependentupon a concentration of the sugar.
 11. The vesicle composition of claim1, a boronic acid derivative moiety conjugated in thephospholipid-polyethylene glycol-boronic acid derivative conjugate beingless cytotoxic and/or less inflammatory than the boronic acid derivativemoiety in unconjugated form.
 12. The vesicle composition of claim 1: thefirst and second phospholipids comprising DPPC; thephospholipid-polyethylene glycol-boronic acid derivative conjugatecomprising a DSPE-PEG-boronic acid derivative conjugate; thephospholipid-polyethylene glycol-glycosyl conjugate comprising aDSPE-PEG-glycosyl conjugate; and the first and second therapeuticcompounds comprising insulin.
 13. The vesicle composition of claim 12,the first liposome comprising one or more of: between about 40 and about70 mole % DPPC; between about 20 and about 50 mole % cholesterol; orbetween about 1 and about 15 mole % DSPE-PEG-boronic acid derivativeconjugate.
 14. The vesicle composition of claim 12, the first liposomecomprising one or more of: about 56.4 mole % DPPC; about 40 mole %cholesterol; or about 3.6 mole % DSPE-PEG-boronic acid derivativeconjugate.
 15. The vesicle composition of claim 12, the second liposomecomprising one or more of: between about 40 and about 70 mole % DPPC;between about 20 and about 50 mole % cholesterol; between about 1 andabout 15 mole % of a DSPE-PEG-glucosyl conjugate; between about 1 andabout 15 mole % of a DSPE-PEG-galactosyl conjugate; or between about 1and about 15 mole % of a DSPE-PEG-mannopyranosidyl conjugate.
 16. Thevesicle composition of claim 12, the second liposome comprising one ormore of: about 56.4 mole % DPPC; about 40 mole % cholesterol; about 1.2mole % of a DSPE-PEG-glucosyl conjugate; about 1.2 mole % of aDSPE-PEG-galactosyl conjugate; or about 1.2 mole % of aDSPE-PEG-mannopyranosidyl conjugate.
 17. The vesicle composition ofclaim 12, at least one of the first and the second liposomes beingconfigured to release an amount of the insulin in response to a presenceof glucose.
 18. A method for treating a subject in need of therapy,comprising: administering to the subject a vesicle compositioncomprising: a first liposome, comprising: a first phospholipid;cholesterol; and a phospholipid-polyethylene glycol-boronic acidderivative conjugate; a second liposome, comprising: a secondphospholipid; cholesterol; and a phospholipid-polyethyleneglycol-glycosyl conjugate, at least one of the first liposome or thesecond liposome respectively encapsulating a first therapeutic compoundor a second therapeutic compound; and the phospholipid-polyethyleneglycol-boronic acid derivative conjugate of the first liposome and thephospholipid-polyethylene glycol-glycosyl conjugate of the secondliposome together being configured to form at least one reversibleboronic-glycosyl crosslink between the first liposome and the secondliposome; and releasing an amount of at least one of the first andsecond therapeutic compounds from at least one of the correspondingfirst and the second liposomes into the subject to provide therapy tothe subject.
 19. The method of claim 18, the administering comprisinginjecting the vesicle composition into the subject.
 20. The method ofclaim 18, the releasing comprising releasing the corresponding amountsof each of the first and second therapeutic compounds from each of thecorresponding first and second liposomes.
 21. The method of claim 18,the releasing comprising releasing the corresponding amounts of each ofthe first and second therapeutic compounds from each of thecorresponding first and second liposomes in response to the presence ofa sugar.
 22. The method of claim 21, the sugar being glucose.
 23. Themethod of claim 18, the releasing comprising releasing the correspondingamounts of each of the first and second therapeutic compounds from eachof the corresponding first and second liposomes, the correspondingamounts being dependent on a concentration of a sugar.
 24. The method ofclaim 18, the releasing comprising releasing insulin from the first andsecond liposomes into the subject.
 25. The method of claim 18, thesubject being in need of treatment for diabetes.